The broad long-term objectives of the proposed research are to develop a new mesenchymal stem-cell (MSC) based therapy for the treatment of pulmonary hypertension. Pulmonary hypertension is a serious, often fatal disease characterized by increased pulmonary arterial pressure, right-heart failure, and remodeling of the pulmonary vascular bed. Although current therapy has prolonged survival, the long-term outcome is not favorable. Gene therapy shows promise, however, in vivo administration of non-viral vectors or viral vectors leads to low level gene transfer, random gene expression, and adverse effects, including inflammation. MSCs are self-renewing adult stem cells from bone marrow, are easily isolated and expanded in culture, and have the potential to differentiate into many cell types. It is our hypothesis that gene-modified MSCs may have a beneficial effect in the treatment of pulmonary hypertension. The results of preliminary experiments in our laboratory show that the administration of gene-modified MSCs and wild-type or unmodified MSCs improve erectile function in the aged rat. The results of preliminary studies also show that intratracheal administration of wild-type MSCs attenuated monocrotaline-induced pulmonary hypertension and restored pulmonary vasodilator responses to acetylcholine, which were impaired by monocrotaline treatment. The experiments proposed in this application will test the hypothesis that intratracheal administration of MSCs or MSCs modified with the eNOS gene or the CGRP gene will have a beneficial effect in monocrotaline-induced pulmonary hypertension in the rat. In order to test this hypothesis, MSCs must be prepared, characterized, gene-modified, and injected into the lung of control rats and rats with monocrotaline-induced pulmonary hypertension. In addition to determining the effect of MSCs and gene-modified MSCs on baseline pulmonary pressures and blood flow, the effects of the MSCs on pulmonary vascular responses to ventilatory hypoxia and vasoactive agonists, including acetylcholine, and the fate of the injected MSCs in the lung will be investigated using histologic and histochemical techniques. Therefore, the first specific aim is to isolate, characterize and gene-modify MSCs from the rat. The second specific aim is to investigate the effect of injected MSCs on pulmonary vascular function and on monocrotaline-induced pulmonary hypertension using newly developed right-heart catheterization techniques. The third specific aim is to study the fate of injected MSCs in the lung using histochemical techniques, the deconvoluted microscope, and localization of the male Y chromosome in the lung of female rats using PCR and FISH methodologies. The results of these studies may lead to new MSC-based therapy for the treatment of pulmonary hypertension.